Translated Abstract
Background: Acute Leukemia is a malignant clonal disease of hematopoietic stem cell disorders in blood system.The affected cells appear uncontrolled proliferation, differentiation disorders and their apoptosis are blocked, the cells accumulate largely in the bone marrow and in other hematopoietic tissue, thereby they inhibite the normal hematopoietic function of bone marrow and infiltrate lymph nodes, spleen, liver and so on. The acute leukemia progresses rapidly and has a high mortality. With the development of modern society,the disease incidence gradually increasing,which has a serious threaten to human health and life. At present, the main treatment of acute leukemia is chemotherapy, supplemented by support treatment.Although the acute leukemia can completely cure by allogeneic hematopoietic stem cell transplantation, but which matching difficult, costing most and the transplantion procedure is dangerous.And the allogeneic hematopoietic stem cell transplantation also has many postoperative complications.So the transplantation is limited by the factors all of above.Chemotherapy is chemotherapy drugs enter into the body to prevent the proliferation of cancer cells, invasion and metastasis . It is a systemic treatment and one of the three major treatment of cancer, and also is the major hematologic malignancies treatment. Doxorubicin is a chemotherapy drug which is widely used in blood system cancers. Doxorubicin (Doxorubicin, adriamycin, ADM), an anti-tumor antibiotic, belonging to anthracyclines / anthraquinones,which can inhibit the synthesis of cellular RNA and DNA, is a cycle non-specific drugs, for S-phase cells are more sensitive. ADM has effects on various growth cycles in the killing of tumor cells. At present, the main treatment of ADM is to combine with other cell cycle-specific drugs induce remission in acute leukemia. Toll-like receptors (TLRs) is a one of the pathogen-associated molecular pattern (PAMP). Previous studies have shown that TLRs mainly expressed in immune tissues and cells, such as macrophages, DCs cells, neutrophils and B cells. However, recent studies have shown, TLRs are also expressed in the tumor cells, and their expression and role are closely relation with tumor genesis and development , which have become a hot research topic. Research shows TLR9 also expressed in tumor cells, U937 cells express too. CpG ODN (CpG oligonucleotide, CpG oligodeoxynucleotides), which is the TLR9s primary ligand. The current study shows :the expression of TLR9 in tumor cells may provide new targets for chemotherapy, radiotherapy, immunotherapy of cancer.
Objective: This study combines ADM with CpG ODN, acting on human acute monocytic leukemia cell line U937, observe and detect whether CpG ODN could enhance the sensitivity of doxorubicin on U937 cells. In order to explore possible ways to enhance the effect of chemotherapy in acute leukemia.
Methods: Reviewed literature to identify the experimental concentrations of CpG ODN. To determine the IC50 of ADM by MTT method,and based on the IC50 to design different concentrations of ADM. To test the cell growth inhibition rate between the alone ADM groups and the ADM combined CpG ODN groups at different concentrations on U937 by MTT method. By light microscopy, Hoechst staining and flow cytometry Annexin V-PI double staining to test the apoptosis induced function between the alone ADM groups and the ADM combined CpG ODN groups at different concentrations on U937 cells. By RT-PCR to detect and analysis apoptosis related genes Bcl-2 and Bax between the alone ADM groups and the ADM combined CpG ODN groups at different concentrations on U937 cell.
Results: checked out relevant literature to determine the concentrations of CpG ODN in experiment is 3ug / ml. tested the IC50 is 0.98 ± 0.05 ug / ml that ADM acted on U937 cells for 24h by MTT. Based on this,we designed 0.25ug / ml, 0.5ug / ml, 1ug / ml, 2ug / ml of ADM performed this experiment. ADM on U937 cell growth significantly inhibited in a dose and time dependent manner.the t test used to test the different concentrations of the alone ADM groups and the ADM combined CpG ODN groups acted on cells after 24h and 36h . There is no significant differences between the two groups of cell growth inhibition rate in the concentration of 0.25ug / ml(P＞ 0.05). But there is significantly different in the concentration of 0.5ug / ml, 1ug / ml, 2ug / ml between the two groups cell growth inhibition rate,and the ADM combined CpG ODN groups cell growth inhibition rate greater than cells alone ADM groups ( P ＜ 0.05). Observed by light microscopy and Hoechst staining, the ADM combined CpG ODN groups acted on U937 cells for 24 hours, with the increasing concentrations of ADM,which significantly increased apoptosis, apoptotic body formation phenomenon than the alone ADM groups. Flow cytometry test resulted Annexin V-PI double staining: the t test was performed at different concentrations of the alone ADM groups and the ADM combined CpG ODN groups on U937 cells after 12h. Concentration were 0.25ug / ml, 0.5ug / ml, 1ug / ml, when 2ug / ml.The early apoptosis rate between the two groups were significantly different, and the ADM combined CpG ODN groups’ rate is greater than t the alone ADM groups（P less than 0.05）. At the same time, all the above concentrations, there is significantly different between the two groups of viable cells, and the ADM combined CpG ODN groups of viable cells were less than the rate of the alone ADM groups（ P less than 0.05）. Compared with the control group, each group of drug-treated U937 cells after 12h, Bax gene mRNA expression levels of ADM combined CpG ODN groups were significantly higher than the alone ADM groups (P ＜0.05). The Bcl-2 mRNA expression changes was no significant difference (P＞ 0.05). Bcl-2 / Bax value decreased that ADM combined CpG ODN group than alone ADM group(P ＜0.05).
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